This project proposes to study the high affinity site for noncompetitive antagonists in the ion channel domain of the nicotinic acetylcholine receptor (AChR) using a strategy based on two recent discoveries from this lab: 1. Certain uncharged, hydrophobic diterpenoids, called cembranoids, noncompetitively inhibit AChR's from muscle, electric organ and nervous system, competing for binding with the channel blocker phencyclidine (PCP). 2. PCP displays higher affinity for the AChR from electrocyte than that from myocyte. This species difference is much smaller for the cembranoid eupalmerin acetate (EUAC). The investigators hypothesize that the affinity difference for PCP between receptors is due to three amino acid substitutions in the M2 transmembrane segment (at positions 6 and 10), while EUAC binds to amino acids in M2 at positions 10 and 13 which overlap the PCP site, but display little difference between the two receptors. Three types of experiments will be carried out to test these hypotheses: 1. Measurements of radioligand binding to Torpedo AChR-rich membranes will be used to determine the potencies of 20 cembranoids as inhibitors of PCP and other noncompetitive antagonists. 2. The cembranoids will be tested as inhibitors of ACh-induced currents for Torpedo AChRs expressed in Xenopus oocytes. 3. Hybrid AChRs constructed from subunits of mouse and Torpedo AChRs, as well as AChRs containing point mutations in the M2 domain, will be expressed in oocytes and assayed to determine PCP and cembranoid binding affinities.